Characterization and Expression of the Human T Cell Receptor-T3 Complex by Monoclonal Antibody F101.01

A murine monoclonal antibody (MoAb) F101.01 reacting with the T cell receptor (TCR)-T3 complex is presented. Immunohistological studies showed that F101.01 specifically stains T-zone lymphocytes in lymph nodes, tonsils, and splenic tissue. Two-colour immunofluorescence and flow cytometry demonstrated co-expression of the antigen defined by F101.01 and the pan-T cell antigens defined by CD2, CD3, CD5, and CD7 antibodies. Cells stained with CD4 and CD8 antibodies were both included in the F101.01-positive population, whereas CD16-positive natural killer cells (NK), B cells (CD19 and CD20), and myeloid cells (CD13 and CD33) were excluded. The target antigen of F101.01 co-modulated with the CD3-defined antigen (T3) and the TCR recognized by the MoAb WT-31. CD3 antibody and WT-31 both blocked binding of F101.01. F101.01 precipitated the TCR-T3 complex from lysates of 125I-labelled peripheral blood mononuclear cells (PBMC) and HPB-ALL, when the lysate was prepared with a detergent (digitonin) that conserves the TCR-T3 complex. FACS analysis of T cells from a patient with a T cell immunodeficiency demonstrated that delta-TCS-1-CD3+CD4+ and delta-TCS-1-CD3+CD8+ cells were brightly F101.01+, whereas a large subpopulation of delta-TCS-1+CD3+CD4-CD8- cells were weakly F101.01+. We conclude that F101.01 recognizes a conformational epitope of the TCR-T3 complex and that it reacts with the alpha beta TCR-T3 and the gamma delta TCR-T3 complexes with different intensities.     

Chlorpromazine inhibits vesiculation, alters phosphoinositide turnover and changes deformability of ATP-depleted RBCs

To delineate further the underlying mechanism by which amphiphilic drugs can modulate vesicle release from human RBCs, we studied the effect of chlorpromazine on erythrocyte vesiculation induced by ATP depletion. This was correlated with turnover of the phosphoinositides as well as RBC deformability during the process since phosphoinositide metabolism may be involved in shape regulation of RBCs. Echinocytic shape transformation and subsequent vesiculation of RBCs, which commonly occur during ATP depletion, were inhibited by chlorpromazine. Furthermore, with a newly developed two-dimensional thin-layer chromatography separation of RBC membrane phospholipids, we showed that chlorpromazine significantly decreased the dephosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP2) in both ATP-depleted RBCs as well as in cells with partly maintained ATP levels. Concomitantly, there was a smaller increase in the relative amount of phosphatidylinositol. In addition, chlorpromazine also inhibited the decreased in RBC deformability as well as the shift of osmotic fragility that occurs during ATP depletion of erythrocytes.     

Soluble interleukin 2 receptors in sera of Japanese patients with adult T cell leukemia mark activity of disease

Serum concentrations of soluble interleukin 2 receptors (sIL 2R) were measured by an enzyme-linked immunosorbent assay (ELISA) in 30 patients with adult T cell leukemia (ATL), in 9 patients with other hematopoietic malignancies, and in 17 asymptomatic individuals seropositive for human T cell leukemia virus type I (HTLV-I). Sixty HTLV-I seronegative, age-matched controls showed a normal range of form 63.2 to 480.8 U/mL. All asymptomatic carriers of HTLV-I had sIL 2R in their sera within the normal range. sIL 2R in sera was not related to the anti-HTLV-I antibody titer. Eleven patients with acute ATL, a clinical phenotype with median survival rate of 4.4 months, had markedly elevated sIL 2R (11,100 to 99,000 U/mL), but eight patients with smoldering ATL had low sIL 2R values (less than 480.8 U/mL) comparable to controls. Eleven patients with chronic ATL had intermediate elevated levels of sIL 2R (480.8 to 37,300.0 U/mL). Serum levels of sIL 2R correlated with the number of ATL cells (r = 0.812) and CD25-positive cells (r = 0.725) circulating in the peripheral blood. Longitudinal studies performed in four patients with ATL showed significant correlation between serum concentration of sIL 2R and activity of the malignancy. These findings suggest that the level of sIL 2R in serum indicated tumor load and, possibly, prognosis.     

Targeted gene transfer to human hematopoietic progenitor cell lines through the c-kit receptor

In this report, we describe a novel gene therapy approach for hematopoietic stem/progenitor cells using a specific receptor-mediated gene transfection procedure to target c-kit+ cell lines. The vector consists of plasmid DNA containing a luciferase reporter gene that is condensed by electrostatic forces with polylysine (PL) covalently linked to streptavidin (binds biotinylated ligand) and PL covalently linked to adenovirus (AD; to achieve endosomal lysis) with the final addition of biotinylated steel factor (SLF-biotin). Targeted transfection of growth factor-dependent hematopoietic progenitor cell lines that express c-kit showed specific luciferase gene expression over cell lines that did not express c-kit. This effect was dependent on the dose of SLF-biotin and was competed by excess SLF or with monoclonal antibodies that recognize c-kit and block the binding of SLF to its receptor. Maximum transfection efficiency (> 90%) requires a 2- hour incubation period of the vector with the cells, and maximum gene expression occurred 30 hours later. Removal of the endosomalytic agent, AD, from the vector resulted in the loss of gene expression. Vector targeting was versatile and could be changed by the addition of other biotinylated ligands. In principle, this vector should be broadly applicable to deliver genes to hematopoietic stem/progenitor cells in vitro and in vivo.     

Activation of the alternative complement pathway with rabbit erythrocytes by circumvention of the regulatory action of endogenous control proteins

Cleavage of C3 by the alternative complement pathway occurs in at least two distinct phases: continuous low grade generation of C3b by the interaction of native C3, B, D, and P, and subsequent amplified cleavage of C3 by the interaction of C3b, B, D, and P which forms the amplification convertase, P,C3b,Bb. Transition to C3b-dependent amplification is necessary to achieve substantial C3 cleavage and is normally limited by the combined action of C3b inactivator (C3bINA) and βlH. An activator of the alternative pathway, such as rabbit erythrocytes (E(r)), provides sites that protect bound C3b and P,C3b,Bb from the action of these regulatory proteins and permits C3b deposited by the low grade fluid phase reaction to assemble a membrane-associated amplification convertase which can deposit additional protected C3b. Under conditions in which the control proteins, C3bINA and β1H, almost completely inactivated C3b bound to sheep erythrocytes (E(s)), which does not activate the alternative pathway, the function of C3b bound to E(r) was diminished by less than one-fifth. Further, the P- stabilized amplification convertase on E(r) was 10-fold less sensitive to β1H-mediated decay-dissociation than the convertase on E(s). The addition of E(r) to a regulated mixture of purified C3, B, D, P, C3bINA, and β1H resulted in amplified inactivation of C3 and B by formation of the amplification convertase on E(r) as indicated by its lysis with subsequent exposure to C3-C9. In contrast, E(s) did not advance the low grade fluid phase inactivation of C3 and B to amplified inactivation and the cell was not converted to an intermediate susceptible to lysis by C3- C9. Since E(r) and E(s) did not differ in their inefficient fixation of C3b generated during an unregulated fluid phase reaction, the activating capacity of E(r) must reside in its protection of bound C3b and P, C3b,Bb from the regulatory proteins rather than in enhanced capacity to bind C3b from the fluid phase. When the reaction is limited to low grade fluid phase turnover, introduction of E(r) but not E(s) results in a 100-fold increase in the deposition of C3b, indicating that surface-dependent activation of the alternative pathway is characterized by efficient deposition of C3b on the initiating surface. Thus, the activating surfaces advance the interaction of the alternative pathway proteins to the amplification phase because of the selective inability of the regulatory proteins to deal with their substrates when deposited on these surfaces and results in a specificity that is not necessarily dependent on adaptive immunity.